Detection of nucleic acid is commonly carried out employing so-called “DNA chips,” which are amenable to high throughput and are more reliable than conventional Southern or Northern blot techniques. Generally, such chips have numerous oligonucleotide fragments (“probes”) of known sequence, and for improved stability it is usually preferred that the probes be either DNA or modified oligonucleotides that are resistant to chemical and enzymatic degradation. During analysis, samples to be investigated are placed in contact with the chip so as to enable hybridization of the probes with the sample DNA or RNA to be analyzed. Next, the hybridized nucleic acid is detected and quantified by some method, usually an optical method using intercalating dyes or other fluorescent labels.
However, such techniques require the use of optical devices and/or fluorescent markers that are very costly. In addition, a quantitative determination of the target DNA present in the sample is difficult. Consequently alternative methods have been studied for detecting chains of DNA on DNA chips, for example of electrochemical type, which make use of the bifunctional compounds.
Bifunctional compounds are currently used for photocleavage of DNA and have two molecular units—one referred to as “intercalating” unit (I) and the other as “active unit” (AD). Generally, the unit I is oxidized by light irradiation so that it transfers one electron to the unit AD. Next, the oxidized unit I is reduced by the DNA before the electron donated to the unit AD returns to the unit I. This mechanism leads to oxidation of the DNA and hence to cleavage thereof as illustrated in FIG. 1 (J. Joseph, et al., J. Phys. Chem. B (2003), volume 107, pages 4444-4450 and references given therein).
It has been found, however, that some bifunctional compounds can also be used for detection of nucleic acids.
Japanese patent No. JP2000-125865 describes a method for detecting a gene by hybridizing it with a DNA probe immobilized on an electrode in presence of an intercalating compound with electrochemical properties, namely, N—N-bis[[4-(3-ferrocenecarboxaminopropyl)piperazinyl]propyl]naphthalen-1,4,5,8-tetracarboxylic acid.
Other examples of intercalating bifunctional compounds for electrochemical detection of chains of double-strand DNA are described in Makoto Takagi, Pure Appl. Chemistry, 2001, vol. 10, pages 1573-1577, where the intercalating unit I is naphthalendiimide and the active unit AD is ferrocene, or in US2002117396, where the compound is N-[3-[4-(3-ferrocenecarboxamidopropyl)piperazinyl]propyl]-1,8-naphthalenimide.
In the bifunctional compounds referred to above, the intercalating unit I can absorb environmental light. Consequently, in appropriate thermodynamic conditions, these compounds can undergo photoinduced processes of electron transfer, which can lead to damage of the DNA as described for the methods of DNA cleavage (J. Joseph, N. V. Eldho, D. Ramaiah, J. Phys. Chem. B (2003), vol. 107, pages 4444-4450 and references contained therein), rendering the DNA unusable for further subsequent analyses or even yielding unreliable analytical results.
Consequently, the known bifunctional compounds must be used in detection applications where appropriate conditions of light are guaranteed and are therefore not suitable for extensive use in industry.
The aim of the present invention is hence to provide light stable bifunctional compounds suitable for use in the analysis of nucleic acids.